1. Field of the Invention
This invention relates to a method, composition and kit for radiolabeling proteins with radioisotopes of technetium and rhenium such as technetium-99m.
2. Description of the Prior Art
The use of radioisotopes to label proteins is well known. These compositions can be used in assays, and can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones and the like. A variety of radioisotopes, including isotopes of iodine, technetium, indium, and rhenium have been used. It is also well known that protein molecules can be tagged or labeled with technetium-99m to form a diagnostic or imaging agent.
Technetium-99m has been utilized to radiolabel proteins, chelating agents, phosphonate bone scanning compositions and the like by a technique which utilizes sodium pertechnetate wherein the technetium initially is in the +7 state. Technetium-99m is generally available only as sodium pertechnetate. The pertechnetate must be contacted with a reducing agent, such as stannous chloride, in order to reduce the technetium to the +3, 4 or +5 oxidation state in the presence of the protein, chelating agent or like substance which is to be radiolabeled. The technetium must be maintained in this reduced state in order to maintain the chemical bond between the technetium molecule and the substrate being radiolabeled. It is also necessary that the technetium be firmly bound to the protein such that the reduced technetium is not transferred to other molecules or other proteins present in the assay, patient's blood or other media in which the radiolabeled substance will be utilized.
Several different methods have been utilized to radiolabel proteins, particularly monoclonal antibodies, with technetium-99m. The methods involve two general approaches. One approach is indirect in which a bifunctional chelating agent is attached to the protein via one functional group and the technetium-99m is attached via the other functional, or chelating, group. This method was introduced by Krejcarek GE and Tucker KL (Biophys Res Comm 77:581-585, 1977) and has been widely employed in many variations using a wide variety of bifunctional chelating agents such as described in the review of Wensel and Meares (Wensel TG and Meares CF: "Bifunctional" Chelating Agents for Binding Metal Ions to Proteins, in Radioimmunoimaging and Radioimmunotherapg, SW Burchiel and BA Rhodes, eds., Elsevier Publishing Co., N.Y., 1983, pp 185-196). Other methods are disclosed by Hnatowich, U.S. Pat. Nos. 4,668,503 and 4,479,930, by Haber et al., U.S. Pat. No. 4,421,735 and by Fritzberg et al., U.S. Pat. No. 4,670,545. The bifunctional chelate methods all present significant limitations, including the complexity of the radiolabeling procedure, the time required to accomplish radiolabeling, and the presence of substances which may affect the protein.
The other general approach is direct labeling. Although several direct methods have been reported, the first direct method capable of providing a sufficiently strong bond between the protein and the technetium-99m for in vivo applications was the direct or pretinning method described in U.S. Pat. No. 4,424,200, entitled "METHOD FOR RADIOLABELING PROTEINS WITH TECHNETIUM-99M", to Crockford and Rhodes. In this method, a single reduction compound, consisting of a solution of stannous chloride and other salts which serves both to reduce the protein, thereby exposing the disulfide bonds, and to reduce the sodium pertechnetate, is used. With this method, many proteins can be successfully radiolabeled with technetium-99m. Several investigators have reported on the use of this method (Rhodes BA et al: "Technetium-99m labeling of murine monoclonal antibody fragments. J Nucl Med 27:685-693, 1986; Som P et al: Radioimmunoimaging of experimental thrombi in dogs using technetium-99m-labeled monoclonal antibody fragments reactive with human platelets. J Nucl Med 27:1315-1320, 1987) and on equivalent methods (Schwarz A and Steinstruaber A: A novel approach to Tc-99m-labeled monoclonal antibodies. J Nucl Med 28:721, 1987; Pak KY et al: A rapid and efficient method for labeling IgG antibodies with Tc-99m and comparison to Tc-99m Fab'. J Nucl Med 30:793, 1989; Granowska M et al: A Tc-99m-labeled monoclonal antibody, PR1A3, for radioimmunoscintigraphy. J Nucl Med 30:748, 1989). In the equivalent methods disulfide reducing agents other than stannous salts were used. Pak et al used dithiothreitol to reduce the disulfide bonds of the antibody; Swartz and Steinsbruaber, and Granowska et al used 2-mercaptoethanol. Also some of these investigators (Swartz and Steinsbruaber, and Granowska et al) reduced the Tc-99m prior to adding it to the reduced antibody, which adds steps to the original procedure.
However, certain proteins, such as some monoclonal antibodies or fragments thereof, cannot be successfully labeled using the previously described direct method or its equivalents. This is primarily due to problems associated with lack of purity of the protein, continued reduction of disulfide bonds in the protein, and the formation of additional reduced protein species prior to admixture with sodium pertechnetate. Fewer stannous ions are needed to reduce pertechnetate than to reduce disulfide bonds in proteins; with some proteins, excess stannous ions cause reduction of disulfide bonds and fragmentation of the protein to less than the desired size. Organic reducing agents such as 2-mercaptoethanol and dithiothreitol can cause aggregation of the reduced antibody or antibody fragments.
Accordingly, it is an object of the present invention to provide an improvement over the previous methods for direct labeling of proteins with technetium-99m, which method will eliminate undesirable fragments or otherwise degraded protein components from the final product.
It is a further object of the present invention to provide a method which will result in increased radiolabeling efficiencies utilizing technetium-99m as the radioisotope.
Another object of the present invention is to provide a method and kit which will permit radiolabeling to be accomplished by the end user using a single vial, containing both reduced antibody and stannous ions, and further containing a means to maintain low quantities of stannous ions while protecting against oxidation loss, which method requires only a single step to accomplish radiolabeling, being the introduction of sodium pertechnetate.
Other objects and the further scope of applicability will become apparent from the detailed description to follow.